The goal of this project is to identify genetic alterations that contribute to the pathogenesis of therapyrelated acute myeloid leukemia and myelodysplastic syndrome (t-AML/t-MDS). These disorders are emerging as a major clinical problem, now accounting for 10-20% of all new cases of AML, with a rising incidence. The available treatments for these diseases have limited efficacy, and the prognosis is very poor, highlighting the need for new therapies. However, the genetic alterations that contribute to t-AML/t- MDS pathogenesis are largely unknown, limiting the development of novel targeted therapies. Major improvements in the clinical management of patients with t-AML/t-MDS are unlikely without a better understanding of the genetic alterations contributing to t-AML/t-MDS susceptibility, transformation, and resistance to chemotherapy. To identify these genetic alterations, the following specific aims are proposed. Aim 1. We will identify novel recurring somatic mutations in t-AML/t-MDS. We propose to sequence at least 150 t-AML/t-MDS genomes over the 5-year grant period. These data will be compared with cfe novo AML/MDS to determine whether prior exposure to chemotherapy or radiotherapy affects the spectrum of somatic mutations found in the tumors. Biological studies will be performed on selected novel recurring mutations to define their contribution to leukemic transformation. Aim 2. We will characterize clonal progression in t-AML/t-MDS. We will take advantage of hematopoietic tissue banked prior to the development of t-AML/t-MDS to address the following questions: 1) does clonal hematopoiesis develop after exposure to intensive chemotherapy; 2) are certain mutations preferentially acquired early during; 3) are mutations that confer resistance to chemotherapy present in hematopoietic stem cell (HSC) clones prior to exposure to chemotherapy; and 4) in cases of t-AML/t-MDS arising after lymphoma, do these tumors share mutations, suggesting a common founder HSC clone? Aim 3. We will determine whether specific small non-coding RNAs (SncRNAs) are consistently dysregulated or mutated in t-AML/t-MDS. We propose to sequence the small RNA transcriptome in t- AML/t-MDS. This data will be use to: 1) identify sncRNAs that are dysregulated in t-AML/t-MDS relative to CD34+ progenitors from healthy donors and leukemic blasts from de novo AML, and 2) to identify novel sncRNAs and determine whether they are targets for somatic mutation in AML. Biological studies will be performed to define the contribution of selected sncRNAs to leukemic transformation.